Reverse phase chromatography is a broadly used purification method for peptides. Several materials to be used as the stationary phase in liquid chromatography comprise a support or carrier and thereon attached functional groups. Such material is also known as bonded phase. A commonly used carrier is silica gel that may suitably be surface modified by means of suitable silanes. The chemistry of silanes with various surfaces is well studied. A general discussion of the reaction of silanes with the surface of silicaceous chromatographic support materials is provided in HPLC Columns: Theory Technology, and Practice, U. D. Neue, Wiley-VCH, Inc., New York (1997). Additional details on the reaction of silanes with porous silicas are disclosed in Characterization and Chemical Modification of the Silica Surface, E. F. Vansant, et al., Elsevier Science B.V. New York (1995). A broad description of the reactions of silanes with a variety of materials is given in Silica Gel and Bonded Phases, Their Production, Properties and Use in LC, R. P. W. Scott, John Wiley & Sons, New York (1993).
The preparation of bonded phases can be made using monofunctional, bifunctional and trifunctional silanes resulting in differently strong bonded layers.
Irrespective of whether mono- bi- or trifunctional silanes are used, it is not possible to react all the silanol groups with the silylating reagents, due to equilibrium reactions and steric hindrance. Almost complete coverage is only obtained if the silylating reagents are horizontally polymerised. It is reported that even with the most aggressive silanization reactions no more than 50% of the silanol groups can be converted to silylated derivatives.
Most commercially available bonded phases are based on monofunctional silanes because of difficulties in the reproducibility in preparing bonded phases using bi- and trifunctional silanes.
Since a significant portion of the original silanol groups remain, these may interact with silanophilic analytes unless they are shielded. A commonly used method to reduce these interactions is the so-called endcapping. Here a small monofunctional silane (e.g. trimethylchloro silane) is reacted with the silicagel surface to provide an additional coverage of the silicagel surface.
U.S. Pat. No. 7,125,488 B2 teaches to solve the hitherto existing problems by providing a polar modified stationary phase material, said material having an inorganic support such as silica gel that is modified with a specific silane providing the polar modification. The polar modifications comprise groups like —O—, —C(O)NH—, —OC(O)NH—, and —(CH2CH2O)n—.
Recently, a strong focus on the development of stationary phases with bimodal chemical surface character has been made. Reversed-phase/ion-exchange (RP/IEX) mixed-mode stationary phases are known and promise great versatility and capability for retaining and separating a variety of charged polar compounds, in addition to more nonpolar, ionic and nonionic analytes. According to the arrangement of functional groups, RP/IEX bimodal columns can be classified into four categories. One type of materials is a mixture of RP and IEX beads. Another type of materials comprises apolar groups and polar groups like amines and carboxylic acids in similar amounts attached to the same bead, a third type of materials comprises apolar chains tipped with polar groups, and a fourth type of mixed-mode materials comprises an anionically or cationically charged group embedded within an apolar chain.
GB 2 074 892 A describes materials with mixed surfaces wherein one kind of ligands is selected from ionic or reversed phase ligands and the second kind is a hydrophilic group like a diol, a diol precursor or an amide.
Also GB 2 431 399 A discloses mixed surfaces with 2 different silyl moieties bonded thereto. Each of these silyl moieties comprises 2 C1-C6 alkyl chains and one further group. In a preferred embodiment said further group is an apolar group on one of the silyl groups and a polar group embedded in apolar chains on the other silyl group.
Also already known are trimodal columns based on nanopolymer silica hybrid technology. Such columns comprise Acclaim® Trinity™ P1 of Dionex. This material consists of high-purity porous spherical silica gel coated with charged (e.g. such as to have cation-exchange functionality) nanopolymer particles and with inner pore areas of the silica gel modified with an organic layer that provides RP and IEX properties.
Other mixed mode materials have been developed and are described under the trademark Kromasil™ (belonging to Akzo Nobel). These one particle-multiple resins stationary phases are described to comprise 50% or 25% of a different functionality like polar functionality (CN, NH2, diol). Although these phases provide better separation in several cases, they are still not satisfactory for a multitude of applications.
CN 101829551 A discloses chromatographic materials bonded with apolar ligands and anionic ligands for use in the separation of melamine from a milk sample and of several phytohormones. The density of anionic groups is disclosed to be 33 to 66%.
Also already known are materials with enhanced ligand density due to horizontal polymerization. These materials have been found to have much higher ligand density than merely grafted materials. First horizontally polymerized materials were described by Fairbank and Wirth, Role of surface-adsorbed water in the horizontal polymerization of trichlorosilanes, Journal of Chromatography A, 830 (1999) 285-291, and further investigated by Li, Carr and Evans, Studies of retention and stability of a horizontally polymerized bonded phase for reversed-phase liquid chromatography, Journal of Chromatography A, 868 (2000) 153-167. Adaptation of this technique to different kinds of ligands, i.e. apolar and polar ligands or apolar and ionic ligands is described in WO 2011/012019 and Wei et al., A new reversed-phase/strong anion-exchange mixed-mode stationary phase based on polar-copolymerized approach and its application in the enrichment of aristolochic acids, Journal of Chromatography A, 1246 (2012) 129-136.
The problem with such horizontally polymerized materials is that a successful production is dependent on the adsorbed water and thus they have to be produced using a quite complicated procedure that renders them very expensive and thereby unsuitable for preparative applications. In addition, although such materials were successfully applied in the analytical enrichment of specific aristolochic acids, they failed in preparative application (Wei et al.)
Since the materials of the state of the art are either not satisfactory with regard to the quality of the separation and/or with regard to the retention time and/or—in particular if desired for preparative applications—with regard to their production costs, there still exists a need for improved materials.